Does bacterial colonization during embryo transfer have an impact on pregnancy rate in ICSI? : Tunisian preliminary results.

INTRODUCTION: Embryos' transfer into the uterine cavity remains a limiting factor in the success of intracytoplasmic sperm injection (ICSI). This study aimed to evaluate the impact of cervico-vaginal colonization on ICSI outcomes. MATERIEL AND METHODS: Longitudinal, prospective and analytical study, conducted from February 1 st to October 31 st, 2017. On the day of embryos' transfer, patients with the following criteria were included: Age ≤ 40, normal uterine ultrasound and hysteroscopy, first or second attempt of ICSI, use of fresh semen and transfer of at least one good quality embryo. Three samples were done for microbiological analysis: vaginal secretions, cervical mucus and the tip of the transfer catheter. RESULTS: The study included 40 patients. Pregnancy rate was 52.5 %. Catheter contamination occurred in nine cases (22.5 %). The most frequently isolated bacteria were Streptococcus anginosus (37 % of cases), Gardnerella vaginalis (27 % of cases) and Streptococcus agalactiae (18 % of cases). In all cases of contamination, the same bacteria were found also in the vagina and cervical mucus. In cases of contamination with Gardnerella vaginalis, the study of vaginal flora showed bacterial vaginosis. No pregnancy was obtained when contamination of the transfer catheter occurred. In the absence of contamination, pregnancy rate was 61.2 % (ρ = 0.003). Both populations (patients with catheter contamination and patients without catheter contamination) were comparable regarding epidemiological characteristics and ICSI attempt data. CONCLUSION: Contamination of the transfer catheter by cervical-vaginal bacteria appears to affect IVF results. Systematic antibiotic prophylaxis could be proposed to reduce this negative impact.

[Bacteriospermia: Effects on semen parameters]. / La bactériospermie : effets sur les paramètres spermatiques.

OBJECTIVES: Bacteriospermia may result in male hypofertlity. Indeed, 60% of patients treated by Assisted Reproductive Techniques had a local infection or inflammation of genital tract. However, direct effects of bacteriospermia on semen parameters remain controversial. The aim of our work was to explore the effect of bacteriospermia on sperm parameters among patients consulting for hypofertility. METHODS: A retrospective comparative study of two groups: a group of 70 patients with bacteriospermia (group 1) and a control group of 70 patients without bacteriospermia (group 2). For these groups, we studied the standard semen parameters (seminal volume and viscosity and spermatozoa count, initial and secondary mobility, vitality and morphology). Comparison of these parameters was made by the Chi2 test and the Fisher test. RESULTS: The mean age of our population was 40.4±6.7 years. There was no significant difference between ejaculate average volume in the 2 groups (group B: 2.78mL versus group A: 2.92mL with P=0.2). Similarly, for the viscosity no difference was noted (P=0,68). The altered parameters in the presence of bacteriospermia were mean concentration and sperm motility (P=0.001 and P=0.049 respectively). The prevalence of secondary asthenospermia was higher in the presence of bacterospermia (P=0.006). No statistically significant differences were observed for morphology and vitality of spermatozoa (P=0.276 and P=0.075 respectively). The leucospermia was associated with bacteriospermia in 10% patients. Ureaplasma urealyticum was the germ most found (45.7%) followed by Streptococcus and Staphylococcacae (20.3% for each), Gram negative rods accounted for 12.9%, while Corynebacterium spp was isolated only in 4,3% patients. U. urealyticum was associated with hypospermia in 33% of cases (P=0.031). Spermatozoa motility was significatively decreased not only in the presence of this bacteria (FT=0.002) but also when cultures were positive for Streptococcus (0.04) and Corynebacterium (P<0.001). Morphological abnormalities were also noticed with Streptococcus with index of teratozoospermia of 46% versus 19% in the 2nd group (P=0.046). CONCLUSION: Bacteriospermia may impair sperm parameters. The treatment of this affection seems to improve sperm fertilizing potential. In addition, it could prevent contamination of culture media.

Solid tumors after chronic lymphocytic leukemia patients: Report of six cases and review of the literature.

INTRODUCTION: Malignancies have been reported to occur with increased frequency in chronic lymphocytic Leukemia (CLL) patients. The aim of this study was to describe which second malignancies occur in patients with CLL, whether these malignancies are related to CLL, its treatment, or both. We also attempt to study factors predicting the development of other malignancies. PATIENTS AND METHODS: Between 1995 and 2009, six cases of CLL associated with solid tumor were diagnosed in Hematology Department of Military Hospital of Tunis. The diagnosis of CLL was made by immunophenotyping of peripheral blood circulating B cells, and the diagnosis of solid tumors was made by biopsy with anatomopathological exam and immunohistochemical study. RESULTS: The mean age of patients was 71 years. Five patients were male. The CLL was classified Stage A in one case, Stage B in three cases and Stage C in two cases. Two patients had abnormal karyotype. Three patients have not received specific treatment for their CLL. Solid tumors were represented by skin cancer in three cases, lung cancer in two cases and breast cancer in one case. The median time between diagnosis of CLL and that of solid tumor was 53 months. CONCLUSION: Patients with CLL have an increased risk of developing a second cancer. Awareness of risk factors could permit early detection.

[Central retinal vein occlusion and internal carotid artery hypoplasia: a case report]. / Occlusion de la veine centrale de la rétine et hypoplasie de l'artère carotide interne: à propos d'un cas.

INTRODUCTION: Retinal vein occlusion is usually encountered in the elderly and is often associated with systemic vascular disease. It is rarer in young subjects and requires a serious etiological search to best adapt the treatment. OBSERVATION: We report the case of a central retinal vein occlusion (CRVO) in a 26-year-old patient who had no pathological history, discovered at a sudden decline of vision in the right eye, reduced to light perception. Fundus examination and retinal angiography showed an edematous CRVO. Radiologic imaging revealed a narrowing of the right carotid canal, probably congenital, and a reduction in the size of the right common carotid artery and internal carotid artery. Progression was favorable without treatment, and visual acuity improved to 8/10. CONCLUSION: The etiological investigation in retinal vein occlusion in young people must be exhaustive. The visual prognosis is variable and may depend on the etiology.

Transfusion-related acute lung injury (TRALI) during remission induction course of acute myeloid leukemia: a possible role for all-transretinoic-acid (ATRA)?

Transfusion-related acute lung injury (TRALI) is a clinical syndrome characterized by sudden onset of respiratory distress due to pulmonary edema during or following transfusion. Two proposed pathophysiologic mechanisms for TRALI were proposed: the antibody hypothesis and the two-event hypothesis. The two-event hypothesis postulates that a pathway to neutrophil activation and aggregation can occur without leukocyte antibodies. We report a case of TRALI occurring during remission induction course of acute myeloid leukemia in a 27-year-old woman who received All-transretinoic-acid (ATRA). We postulate that ATRA may have played a role in this life-threatening complication by priming neutrophil and enhancing their adherence and their activation in the pulmonary endothelium. TRALI improved with non-invasive ventilation support and use of high dose corticosteroids.

Similar pathological effects of sympathectomy and hypercholesterolemia on arterial smooth muscle cells and fibroblasts.

In a previous study, we showed that after sympathectomy, the femoral (FA) but not the basilar (BA) artery from non-pathological rabbits manifests migration of adventitial fibroblasts (FBs) into the media and loss of medial smooth muscle cells (SMCs). The aim of the present study was to verify whether similar behaviour of arteries occurred in the pathological context of atherosclerosis. Thus, similar experiments were conducted on hypercholesterolemic rabbits, which were chemically sympathectomized with 6-hydroxydopamine (n=4) or treated with vehicle for control (n=5). Cross-sections of BA and FA were immunolabelled for five markers of phenotypic modulation of vascular SMCs and FBs: vimentin, desmin, alpha-smooth muscle actin, beta-isoform of actin, and h-caldesmon and examined using a confocal microscope. Also, 3D images were constructed and morphometric analysis performed using image analysis software. Both intact and sympathectomized BA and FA developed atherosclerotic plaques, but the thickening of the intima was more advanced in sympathectomized animals, as judged by increased plaque frequency and by the phenotypic modulation of SMCs in the intima. Our results show that in the media of FAs hypercholesterolemia induces changes similar to those observed in sympathectomized rabbits in non-pathological conditions, i.e., migration of adventitial FBs to the media and loss of medial SMCs. These latter changes, which can be ascribed to pathological events, were accentuated after sympathectomy in the hypercholesterolemic rabbits. The present study reveals that pathological events, including migration and phenotypic modulation of vascular FBs and loss of SMCs, may be under the influence of sympathetic nerves.

Differing influence of sympathectomy on smooth muscle cells and fibroblasts in cerebral and peripheral muscular arteries.

In the present study, we examined the effect of sympathectomy on the distribution and the relative expression of cytoskeletal proteins used as markers of phenotypic modulation of vascular smooth muscle cells (SMCs) and myofibroblasts (MFBs) in rabbit femoral (FA) and basilar (BA) arteries. Adult rabbits were treated either with repeated 6-hydroxydopamine (6-OHDA) for sympathectomy or with vehicle for control. Cross sections taken from sympathectomized and control arteries 79 days later were immunolabelled for vimentin, desmin, alpha-smooth muscle actin (alpha-SM actin), beta-isoform of actin and h-caldesmon. The distribution of these proteins and the intensity of fluorescent labelled SMCs were examined under a confocal microscope. In the sympathectomized BA, there was no change for desmin, vimentin and h-caldesmon expression, but the expression of both alpha-SM actin and the beta-isoform was significantly higher (+19% and +30%, respectively). In the sympathectomized FA, the expression of the alpha- and beta-isoforms of actin remained unchanged, whereas those of desmin and vimentin were significantly higher (+35% and 17%, respectively) and h-caldesmon expression was lowered by 13%. In contrast to intact FAs, the external layers of sympathectomized FAs revealed migration of fibroblasts from the adventitia and death of SMCs. These results strongly suggest that sympathetic nerves intervene in the cytoskeletal protein remodelling through phenotypic modulation of both SMCs and MFBs during post-natal development, and in pathologies involving similar phenomena, such as atherosclerosis.

Confocal microscopic evidence of decreased alpha-actin expression within rabbit cerebral artery smooth muscle cells after subarachnoid haemorrhage.

Our objective was to determine whether subarachnoid haemorrhage modifies cerebral artery smooth muscle cell phenotype and the contractile protein alpha-actin measured 7 days after haemorrhage. We used a rabbit subarachnoid haemorrhage model and immunofluorescence labelling of alpha-smooth muscle actin, vimentin and desmin. The paired comparison between the haemorrhage and sham rabbits was performed using confocal laser-scanning microscopy. We found in the haemorrhage group significantly less intense alpha-actin immunostaining (p = 0.036) and more intense vimentin immunostaining (p = 0.043) but no significant change in the intensity of desmin staining. Our results indicate an absolute decrease after subarachnoid haemorrhage in the amount of functional alpha-actin and in the light of the literature may suggest a certain degree of dedifferentiation of smooth muscle cells in the cerebral artery wall.

Dynamic in vivo measurement of erythrocyte velocity and flow in capillaries and of microvessel diameter in the rat brain by confocal laser microscopy.

A new method for studying brain microcirculation is described. Both fluorescently labeled erythrocytes and plasma were visualized on-line through a closed cranial window in anesthetized rats, using laser-scanning two-dimension confocal microscopy. Video images of capillaries, arterioles, and venules were digitized off-line to measure microvessel diameter and labeled erythrocyte flow and velocity in parenchymal capillaries up to 200 microm beneath the brain surface. The method was used to analyze the rapid adaptation of microcirculation to a brief decrease in perfusion pressure. Twenty-second periods of forebrain ischemia were induced using the tour-vessel occlusion model in eight rats. EEG, arterial blood pressure, and body temperature were continuously controlled. In all conditions, labeled erythrocyte flow and velocity were both very heterogeneous in capillaries. During ischemia, capillary perfusion was close to 0, but a low blood flow persisted in arterioles and venules, while EEG was flattening. The arteriole and venule diameter did not significantly change. At the unclamping of carotid arteries, there was an instantaneous increase (by about 150%) of arteriole diameter. Capillary erythrocyte flow and velocity increased within 5 seconds, up to, respectively, 346 +/- 229% and 233 +/- 156% of their basal value. No capillary recruitment of erythrocytes was detected. All variables returned to their basal levels within less than 100 seconds after declamping. The data are discussed in terms of a possible involvement of shear stress in the reperfusion period.

Structural organization of the perivascular astrocyte endfeet and their relationship with the endothelial glucose transporter: a confocal microscopy study.

Despite the increasing evidence for a prominent role played by the perivascular endfeet of astrocytes in the functional metabolic coupling between astrocytes and neurons, a clear picture of their spatial organization is still lacking. To examine the three-dimensional structure of the astrocyte endfeet and their relationships with the endothelial cells, coronal rat brain sections immunolabeled for the two astroglial markers [glial fibrillary acidic protein (GFAP)/S-100beta] and the endothelial glucose transporter (GLUT1) were analyzed under the confocal microscope. Double immunolabeling of GFAP and S-100beta showed numerous well-defined astrocytes sending one or more endfeet to the vasculature. Examination of GFAP immunolabeling at higher magnification showed that these endfeet consist of well-defined rosette-like structures lying on the vessel wall. Double immunostaining of GFAP and GLUT1 showed that the endothelial cells were the main targets of these repeated geometrical units formed by the astrocyte endfeet. When three-dimensional images were reconstructed, obvious privileged anatomical relationships were observed between endfeet and individual endothelial cells. These anatomical data provide strong support for the involvement of astrocytes in cerebral metabolic coupling. The finger-like appearance of astrocyte endfeet could allow direct metabolic exchanges between intracerebral vessels and non-glial elements such as nerve terminals.

Effect of sympathectomy on the phenotype of smooth muscle cells of middle cerebral and ear arteries of hyperlipidaemic rabbits.

This study was carried out to determine whether sympathectomy influences the phenotypic modulation of smooth muscle cells in the peripheral and cerebral arteries of heritable hyperlipidaemic rabbits. Unilateral superior cervical ganglionectomy (common origin of innervation to the middle cerebral artery and the central ear artery) was performed on four Watanabe heritable hyperlipidaemic rabbits. Cross-sections of the ipsi- (sympathectomized) and the contralateral (intact) cerebral and ear arteries were prepared 2 months later and labelled with monoclonal antibodies against vimentin and desmin, two markers of the differentiation of smooth muscle cells, and alpha-smooth muscle actin, a marker of these cells. Sections from control and sympathectomized arteries were analysed with a confocal laser scanning microscope. Compared with contralateral intact ear arteries, the sympathectomized ear artery developed a thickened intima with dedifferentiated smooth muscle cells, expressing alpha-smooth muscle actin but no desmin, whereas the middle cerebral artery remained unchanged. These results suggest that sympathectomy may favour the progression of atherosclerosis in peripheral but not in cerebral arteries of Watanabe heritable hyperlipidaemic rabbits.

Differential processes of vascular smooth muscle cell differentiation within elastic and muscular arteries of rats and rabbits: an immunofluorescence study of desmin and vimentin distribution.

Two main populations of smooth muscle cells exist in the arterial media of adult mammals with respect to expression of two intermediate filament proteins: vimentin-positive/desmin-negative cells (V+/D-) and vimentin-positive/desmin-positive ones (V+/D+). However, it is still not understood how this phenotypic diversity is established. Since the proportion and the distribution patterns of the two muscle cell populations depend both on the type of blood vessel and the species examined, the aim of the present study was to determine and to compare their developmental origin in various artery segments from two different species. Using confocal scanning laser microscopy and sections stained by means of immunofluorescence, the distribution patterns of desmin and vimentin were compared in transverse sections of thoracic and abdominal aortas (elastic arteries) and of the femoral artery (muscular artery) of newborn and adult rats (n = 12) and rabbits (n = 12). The comparison of sections labelled with specific antibodies showed the existence of a subpopulation of smooth muscle cells in the aortas, but not in the femoral artery, which expressed desmin in newborns but not in adults. These data suggest that the phenotype of smooth muscle cells in elastic arteries but not in muscular arteries is modulated during development.

Microvessels isolated from brain: localization of muscarinic sites by radioligand binding and immunofluorescent techniques.

The present investigation was carried out to determine the extent to which muscarinic acetylcholine receptors (mAChRs) in vascular and perivascular structures were colocalized with glial fibrillary acidic protein (GFAP)-positive structures. To this aim, an immunocytochemical approach on free-floating cryosections and isolated microvessels obtained from rat brain was performed to study the possible colocalization of immunostaining with the anti-mAChR protein antibody (M35) and an anti-GFAP antibody. Double-labeling experiments were carried out by fluorescent techniques. Confocal microscopic observations of GFAP and M35 immunoreactivities on free-floating sections showed a high degree of colocalization on astrocyte processes associated with large vessels or capillaries. This pattern suggests that muscarinic receptors are associated with astrocytic endfeet. Confocal microscopic observations of immunoreactivity from isolated cerebral microvessels strengthen this conclusion since double-labeling of M35 and GFAP showed that perivascular astrocytic structures remained attached to the isolated microvessels and were present on vascular segments showing M35 immunoreactivity. In another set of experiments, the specific binding of [3H]quinuclidinylbenzylate ([3H]QNB) to isolated microvessel membrane preparations from cerebral cortex, caudate nucleus, thalamus, and cerebellum showed that a constant binding yield (20% in bovine and 40% in rat) was observed for microvessels compared with the corresponding brain region. According to our immunocytochemical results, the astrocytic membrane remaining attached to microvessels may account for the majority of the muscarinic binding to isolated microvessels. [3H]QNB binding values found in isolated microvessels cannot therefore be considered as artifacts without any link with vascular function. Taken together, the present study strengthens the idea that the muscarinic receptors may be implicated in the functional relationship between glial and vascular structures.

Chemical sympathectomy favours vimentin expression in arterial smooth muscle cells of young rats.

The aim of the present study was to determine whether sympathectomy could influence the relative expression of two intermediate filament proteins, desmin and vimentin, two markers of differentiation, in arterial smooth muscle cells of the young rat. Newborn rats were treated with either repeated guanethidine or saline injections. Sections of the abdominal aorta, the femoral artery, the basilar and the middle cerebral arteries were processed simultaneously for immunofluorescence with monoclonal antibodies against desmin and vimentin and were then examined under either a conventional or a confocal laser-scanning microscope. In both cases, the mean optical density of the muscle layer staining was estimated by computerized image analysis. All artery sections from guanethidine-treated animals showed a significantly higher vimentin expression (108-119% of control values). Desmin expression was not significantly different except in the femoral artery after sympathectomy, where it was decreased by 6%. The relative increase of vimentin expression in sympathectomized blood vessels observed in the present study directly confirms previous morphometric and ultrastructural studies indicating that the sympathetic innervation of cerebral and peripheral blood vessels influences the phenotypic features of smooth muscle cells during post-natal development.

Substance P, calcitonin gene-related peptide, and capsaicin release serotonin from cerebrovascular mast cells.

Rabbit leptomeningeal arteries contain granular cells resembling mast cells that frequently contact autonomic and sensory nerve profiles. In the present in vitro study, we determined whether these cells could be stimulated by substance P (SP) and calcitonin gene-related peptide (CGRP), which are stored and released by sensory C fibers. Immunohistochemistry of the middle cerebral artery showed that 5-HT was stored only in mast cell-like granules. This pool of 5-HT decreased in a dose-dependent manner when exogenous SP and CGRP were added to the incubation solution or when endogenous neuropeptides were released from nerve terminals by capsaicin. The simultaneous administration of CGRP and SP induced a dramatic exocytosis and a 5-HT release significantly greater than the sum of the individual effects of the two neuropeptides. We conclude that, as in classical connective tissue mast cells, the amine content of these granular cells can be released by a degranulation process induced by neuropeptides. The effects of capsaicin suggest that this phenomenon can be triggered by axon reflex of C fibers. The data also provide the first evidence of a synergistic action of SP and CGRP on mast cell degranulation.